The present invention provides microfluidic apparatus, methods and integrated systems for the separation and analysis of reaction components, fluid velocities, component velocities and reaction rates. Exemplary software is provided.
There exists a need for assay methods and associated equipment and devices that are capable of performing repeated, accurate assays that operate at very small volumes. U.S. Ser. No. 08/761,575 entitled xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d by Parce et al. (see also, U.S. Ser. No. 08/881,696) provides pioneering technology related to microscale fluidic devices, including electrokinetic devices. The devices are generally suitable for assays relating to the interaction of biological and chemical species, including enzymes and substrates, ligands and ligand binders, receptors and ligands, antibodies and antibody ligands, as well as many other assays.
In the electrokinetic devices provided by Parce et al., an appropriate fluid is placed in a microchannel etched into a substrate having functional groups present at the surface. The groups ionize when the surface is contacted with an aqueous solution. For example, where the surface of the channel includes hydroxyl functional groups at the surface, protons can leave the surface of the channel and enter the fluid. Under such conditions, the surface possesses a net negative charge, whereas the fluid will possess an excess of protons, or positive charge, particularly localized near the interface between the channel surface and the fluid. By applying an electric field along the length of the channel, cations will flow toward the negative electrode. Movement of the positively charged species in the fluid pulls the solvent with them.
Improved methods and devices for monitoring reactions between chemical or biological species would be desirable. Electrokinetic microfluidic devices and assays using such devices are particularly desirable, due to the general adaptability of electrokinetic movement of small volumes of fluids to high throughput assay systems. The present invention fulfills these and a variety of other needs.
It has now been discovered that accurate determination of the reaction rate of a reaction conducted in a microscale fluidic device is facilitated by consideration of the velocity of the components in the reaction. In a microscale system in which the flux of reactants and reaction products is conserved, the velocity of at least one reactant or product is determined and the concentration of a reaction product is measured or calculated, facilitating determination of the reaction rate.
The concentration of products and reactants is typically measured at a selected position on the microscale fluidic device, e.g., spectrophotometrically, radioscopically, electrochemically, or optically. Velocity rates are optionally determined by measuring the speed of a component in a portion of the microscale fluidic device over time, or are determined by consideration of the parameters influencing velocity, e.g., the charge and mass of the component in an electric field. As described herein, methods of determining velocities are also provided in a constant flux state by indirect measurements, e.g., the velocity of a reactant or product can be determined by measuring a different reactant or product. Thus, any or all reactants or product velocities can be observed or determined. Velocity markers are also optionally used to approximate velocity. In one series of embodiments, electrokinetic devices and fluid injection schemes are described which self-correct for velocity effects on fluids.
A variety of reactants and products are assessed by these methods, including ligand and ligand binders such as an antibody and an antibody ligand, a receptor and a receptor ligand, biotin and avidin, proteins and complementary binding proteins, carbohydrates and carbohydrate binding moieties, nucleic acids, etc. Reactions which are monitored are fluorogenic or non-fluorogenic. A variety of microscale apparatus are adaptable to the methods such as microvalve and micropump arrangements, and particularly electrokinetic devices and the like. Multiple reactants and products are optionally assessed by serial or simultaneous detection methods or a combination thereof.
In one preferred class of embodiments, the microscale fluidic device provides for electrokinetic movement of reactants and products along a microfluidic channel. An electrokinetic microfluidic device is provided, having a microfluidic channel. An electric field is applied along the length of the microchannel, thereby causing charged species such as reactants, solvent molecules and products to move along the length of the channel due to electrophoretic flow, as well as by electroosmotic flow of the solvent in the channel. A first reaction component having a first charge mass ratio (CM1) and a first velocity (U1) is contacted to a second reaction component having a second charge mass ratio (CM2) and a second velocity (U2) in the microchannel, thereby permitting formation of a reaction product with a third charge mass ratio (CMp) and a third velocity (Up). Additional reaction components and products are optionally provided and assessed for velocities and concentrations. In one embodiment, a reactant can have a velocity of zero, e.g., because it is fixed to a substrate of the detection apparatus. However, the more typical case is for flowing reactants, where all reactants and products are flowing in channels of the system. Typically, the product has a velocity different from one or more reactants in the system.
Apparatus for practicing the methods of the invention are provided. For example, a microfluidic detection apparatus for determining the rate of formation of a moving analyte on an electrokinetic microfluidic substrate is provided. The apparatus has a microfluidic substrate holder for receiving a microfludic substrate during operation of the apparatus, having a microfluidic substrate viewing region. An analyte detector such as a phototube, photodiode, a charge coupled device, a camera, a microscope, a spectrophotometer, or the like is mounted proximal to the substrate viewing region to detect the moving analyte in a portion of the substrate viewing region. A computer operably linked to the analyte detector is provided. The computer determines the rate of formation of the analyte, correcting for the effects of the motion of the analyte, e.g., by determining or collating the velocities of one or more components and the concentrations of one or more components and calculating the rate of formation of one or more components, correcting for the velocity of the components. In preferred embodiments, the apparatus also includes an electrokinetic fluid direction system for moving fluids in the microfluidic substrate, such as one or more electrodes which fit into wells of the substrate, operably coupled to one or more electrical power supply.
Electrokinetic microfluidic devices are also provided. The devices have a substrate or body with a top portion, a bottom portion and an interior portion. The interior portion has at least two intersecting channels, with at least one of the two intersecting channels having at least one cross sectional dimension between about 0.1 xcexcm and 500 xcexcm. The device has an electrokinetic fluid direction system for moving an analyte through at least one of the two intersecting channels, a detection zone for detecting the analyte within at least one of the two intersecting channels, when the analyte is in motion, and a data detection device for detecting the analyte in the detection zone. A data analyzer which determines a rate of formation of the analyte in motion, such as a computer, is operably connected to the microfluidic device, e.g., with cables to the data detection device, or by recording data on the data collection device and transporting the recorded data (e.g., on a computer-readable storage medium) to the computer. Typically, the computer has appropriate software for determining reaction rates and other related information.
In one embodiment, at least two intersecting channels are etched in a top surface of the bottom portion, with the top portion being fused to the top surface of the bottom portion, thereby forming the interior portion disposed between the top portion and the top surface of the bottom portion. When heat lamination of glass or polymeric surfaces is performed, the glass or polymer fuses, typically with no seam existing between the top and bottom portion of the resulting microfluidic chip. In one preferred embodiment, the top portion of the device has a plurality of wells in fluid communication with the electrokinetic fluid direction system comprising an electrode adapted to fit into at least two of the plurality of wells. By applying an electric current with the electrode, solvent and analyte molecules are moved through the channels.